The present invention relates to synthetic peptides and to pharmaceutical compositions comprising them for the diagnosis and treatment of anti-phospholipid syndrome.
ABBREVIATIONS: AFC: antibody-forming cells; APS: anti-phospholipid syndrome; HUVEC: human umbilical vein endothelial cells; mAb: monoclonal antibody; MAP: multiple antigenic peptide; PBL: peripheral blood lymphocytes; SLE: systemic lupus erythematosus; St: streptavidin; xcex22GPI: beta-2-glycoprotein 1.
Autoimmune diseases are disorders in which the immune system produces autoantibodies directed against an endogenous antigen, with consequent injury to tissues. These self antigens, called also autoantigens, despite being normal tissue constituents, are the target of a humoral or cell-mediated immune response that characterizes the autoimmune disease.
Several connective tissue disorders including vascular diseases, such as vasculitis, systemic lupus erythematosus (SLE), and polymyositis, neurologic diseases such as multiple sclerosis and myasthenia gravis, and hematologic diseases such as idiopathic thrombocytopenia purpura (ITP) and anti-phospholipid syndrome (APS) seem to be caused by an autoimmune reaction. For some of these disorders, the self antigen has been identified and/or pathogenic autoantibodies have been identified and isolated.
No specific drugs exist nowadays for the treatment of autoimmune diseases and patients are treated with anti-inflammatory drugs such as corticosteroids and/or immunosuppressive drugs. All research being carried out in this field is directed to the development of drugs specific for each disease.
Anti-phospholipid antibodies have been associated with a variety of clinical phenomena, including arterial and venous thrombosis, thrombocytopenia, and obstetric complications. The term xe2x80x9canti-phospholipid syndromexe2x80x9d is used to link a variety of thrombotic events to antibodies against specific proteins involved in blood coagulation. Thrombotic events are reported in approximately 30% of patients with anti-phospholipid antibodies, with an overall incidence of 2.5% patients/year. Deep vein thrombosis of the legs and/or thrombotic events, and cerebral arterial thrombosis are the most common arterial complications. Obstetric complications include recurrent spontaneous miscarriages, fetal deaths, or fetal growth retardations. Women with anti-phospholipid antibodies are particularly prone to second or third trimester fetal death.
The anti-phospholipid syndrome (APS) is characterized by the presence of high titers of anti-cardiolipin and/or anti-xcex22GPI (beta-2-glycoprotein 1) antibodies which might have lupus anti-coagulant activity leading to thromboembolic phenomena, thrombocytopenia, recurrent fetal loss, as well as other multisystemic involvements. APS can emerge as a primary syndrome or as secondary syndrome to SLE (Hughes et al., 1986; McNeil et al., 1991).
Anti-xcex22GPI antibodies bind anionic phospholipids through the xcex22GPI molecule (McNeil et al., 1990; Igarashi et al., 1996). xcex22GPI is the target antigen for the autoimmune anti-xcex22GPI antibodies previously entitled xe2x80x98anti-cardiolipin/anti-phospholipid xcex22GPI dependent antibodiesxe2x80x99. xcex22GPI (50KD), initially described by Schultze et al. (1961), is composed of five respective consensus (xe2x80x98sushixe2x80x99 like) repeats (Kandiah and Krilis, 1994). xcex22GPI binds negatively charged phospholipids through a lysine-rich locus (Cys281-Cys288) located in the fifth domain (Hunt and Krilis, 1994) and possesses several in vitro properties which define it as an anticoagulant, i.e., it causes inhibition of prothrombinase activity, ADP-induced platelet aggregation, platelet factor IX production (Sheng et al., 1996). Employing site-directed mutagenesis of recombinant human xcex22GPI, a cluster of lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity was identified (Sheng et al., 1996).
The anti-xcex22GPI antibodies have been considered to exert a direct pathogenic effect by interfering with hemostatic reactions occurring on the surface of platelets or vascular endothelial cells (Shi et al, 1993; Simantov et al., 1995). Passive transfer of these antibodies into naive mice or mice prone to develop APS, resulted in induction of experimental APS in mice (Blank et al., 1991). It has been shown recently (Del Papa et al., 1997; George et al., 1998) that human polyclonal and monoclonal anti-xcex22GPI antibodies react in vitro with endothelial cells through adherent xcex22GPI and induce differential endothelial cell activation. It is not clear to which epitopes on the xcex22GPI molecule these anti-xcex22GPI antibodies are directed, and the correlation to their biological activity.
Attempts have been made to find peptides that could mimic the self antigen-epitope and would inhibit the autoantibody/self antigen binding and consequent injury to the tissue. Thus, recently, peptides selected from phage-epitope libraries through binding to pathogenic monoclonal autoantibodies were shown to provide a surrogate antigen or mimotope that inhibits binding to the original antigen. Such peptides reflect the sequence or conformation of the antigen-binding site, and the fine specificity of the autoantibodies to the protein and non-protein, e.g. polysaccharides or dsDNA, antigens (Scott and Smith, 1990; Scott et al., 1992; Yayon et al., 1993).
It is an object of the present invention to provide means for diagnosis and specific treatment of the autoimmune disorder anti-phospholipid syndrome (APS).
A further object of the invention is to provide means for inactivating B-cells responsible for the production of autoantibodies appearing in APS patients.
The present invention relates to synthetic peptides suitable for the diagnosis and treatment of APS, more particularly to synthetic peptides and derivatives thereof capable of inhibiting the biological activity of anti-xcex22GPI mabs in vitro. and of inhibiting induction of experimental APS in mice by anti-xcex22GPI mAbs.
In a preferred embodiment, the peptides of the invention and their derivatives are selected from the group consisting of:
(i) a peptide of at least 4 amino acid residues comprising a sequence selected from:
(a) Thr-Pro-Arg-Val
(b) Lys-Ala-Thr-Phe
(c) Leu-Arg-Val-Tyr
(ii) a cyclic derivative of a peptide of (i);
(iii) a peptide according to (i) or (ii) in which one or more amino acid residues have been replaced by the corresponding D-isomer or by a non-natural amino acid residue;
(iv) a chemical derivative of a peptide according to (i)-(iii);
(v) a multichain peptide-oligomer/polymer conjugate comprising two or more of the same or different peptides or peptide derivatives (i) to (iv) attached to a native or synthetic multifunctional oligomeric or macromolecular backbone; and
(vi) a multiple antigen peptide (MAP) in which two to eight same or different peptides or peptide derivatives (i) to (iv) are attached to a diaminoalkanoic acid core.
In one embodiment, a peptide according to (i)(a) above has a sequence selected from:
(a1) Leu-Lys-Thr-Pro-Arg-Val
(a2) Lys-Thr-Pro-Arg-Val-Thr
(A) Asn-Leu-Lys-Thr-Pro-Arg-Val-Gly-Gly
In another embodiment, a peptide according to (i)(b) above has a sequence selected from:
(b1) Lys-Asp-Lys-Ala-Thr-Phe
(B) Lys-Asp-Lys-Ala-Thr-Phe-Gly-Thr-His-Asp-Gly
In a further embodiment, a peptide according to (i)(c) above has a sequence selected from:
(c1) Thr-Leu-Arg-Val-Tyr-Lys
(c2) Thr-Lys-Leu-Arg-Val-Tyr
(c3) Thr-Leu-Leu-Arg-Val-Tyr
(C) Cys-Ala-Thr-Leu-Arg-Val-Tyr-Lys-Gly-Gly
(i) a peptide of at least 4 amino acid residues comprising a sequence selected from:
(a) Thr-Pro-Arg-Val (residues 1-4 of SEQ ID NC:1)
(b) Lys-Ala-Thr-Phe (residues 3-6 of SEQ ID NO:4)
(c) Leu-Arg-Val-Tyr (residues 4-7 of SEQ ID NO:7)
It has further been found according to the present invention that when a number of the same or different peptides or peptide derivatives (i) to (iv) which recognize and bind to autoantibodies secreted by specific B cells in APS are attached to a multifunctional oligomolecular or macromolecular backbone, the resulting molecule according to (v) above (hereinafter designated peptide xe2x80x9cdimerxe2x80x9d, xe2x80x9ctetramerxe2x80x9d, etc.) is capable of inhibiting the production of said autoantibodies by said specific B cells.
The multifunctional oligomolecular or macromolecular backbone may be derived from a native oligomolecular or macromolecular compound such as proteins, oligopeptides, oligosaccharides and oligonucleotides. Suitable proteins are for example albumins, globulins, avidin, and streptavidin.
In one embodiment, a peptide according to (i)(a) above has a sequence selected from:
(a1) Leu Lys Thr Pro Arg Val (SEQ ID NO:1)
(a2) Lys Thr Pro Arg Val Thr (SEQ ID NO:2)
(A) Asn Thr Leu Lys Thr Pro Arg Val Gly Gly (SEQ ID NO:3)
In another embodiment, a peptide according to (i)(b) above has a sequence selected from:
(b1) Lys Asp Lys Ala Thr Phe (residues 1-6 of SEQ ID NO:4)
(B) Lys Asp Lys Ala Thr Phe Gly Thr His Asp Gly (SEQ ID NO:4)
In a further embodiment, a peptide according to (i)(c) above has a sequence selected from:
(c1) Thr Leu Arg Val Tyr Lys (residues 3-8 of SEQ ID NO:7)
(c2) Thr Lys Leu Arg Val Tyr (SEQ ID NO:5)
(c3) Thr Leu Leu Arg Val Tyr (SEQ ID NO:6)
(C) Cys Ala Thr Leu Arg Val Tyr Lys Gly Gly (SEQ ID NO:7)
The invention further provides pharmaceutical compositions comprising a peptide or a derivative thereof of the invention and a pharmaceutically acceptable carrier for the treatment of APS, a method for the treatment of APS which comprises administering to a patient in need thereof an effective amount of a peptide or peptide derivative of the invention, and a method for inactivating B cells or killing the specific B cells responsible for the production of autoantibodies appearing in a patient suffering from APS which comprises administering to said patient an effective amount of a multichain peptide or a multiple antigen peptide of the invention.
The invention further provides diagnostic kits comprising one or more peptides or derivatives thereof of the invention, representing target epitopes for the diagnosis of anti-phospholipid antibodies with different pathogenic biofunctions, which may correlate either with pregnancy complications, thrombosis, or coagulation dysregulation. These kits will allow a quicker diagnosis of the presence of specific autoantibodies, and possibility to provide a more specific treatment for patients with anti-phospholipid syndrome.
In the description hereinafter the following terminology will be used:
Peptide monomer: a single inhibitory peptide A, B, C or irrelevant peptide D.
Peptide dimer: a conjugate of two molecules of biotinylated peptide monomer A, B, C, or D, with one molecule of streptavidin, herein St-diA, St-diB, St-diC, or St-diD.
Peptide tetramer: a conjugate of four molecules of biotinylated peptide monomer A, B, C, or D, with one molecule of streptavidin, herein St-tetraA, St-tetraB, St-tetraC, or St-tetraD.
Divalent peptide: an MAP obtained by attaching 2 molecules of peptide A, B, C or D to 2 molecules of FmocLys(Fmoc)-OH via Gly and/or Ala as spacer. wherein the protective Fmoc (9-fluorenylmethyloxycarbonyl) group is removed during the process of attaching the desired peptide, thus resulting in divalent peptides represented as Lys(xcex1,xcex5-diA), Lys(xcex1,xcex5-diB), Lys(xcex1,xcex5-diC), or Lys(xcex1,xcex5-diD), herein xe2x80x9cdivalent A, B, C or Dxe2x80x9d.
Tetravalent peptide: an MAP obtained by attaching 4 molecules of peptide A, B, C or D to 4 molecules of FmocLys(Fmoc)-OH via Gly and/or Ala as spacer, thus resulting in tetravalent peptides represented as (di-xcex1,xcex5-Lys)2Lys(tetraA), (di-xcex1,xcex5-Lys)2Lys(tetraB), (di-xcex1,xcex5-Lys)2Lys(tetraC), (di-xcex1,xcex5-Lys)2Lys(tetraD), herein xe2x80x9ctetravalent A, B, C or Dxe2x80x9d.